Bistability in the Rac1 PAK and RhoA Signaling Network Drives Actin Cytoskeleton Dynamics and Cell Motility Switches

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe members of the Rho family of small guanosine triphosphatase (GTPases), RhoA and Rac1, play crucial roles in a range of cellular functions, including the regulation of the actin cytoskeleton, cell polarity and migration, gene expression, and cell proliferation (, ). Rho GTPases function as molecular switches, cycling between inactive guanosine diphosphate (GDP)-bound (“off”) and active GTP-bound (“on”) states. In their “on” state, Rho GTPases can bind downstream effector proteins, initiating signaling through multiple pathways. The GTPase activation-deactivation cycle is tightly controlled by two opposing enzyme groups, (1) guanine exchange factors (GEFs), which facilitate switching from GDP to guanosine triphosphate (GTP), and (2) GTPase-activating proteins (GAPs), which stimulate GTP to GDP hydrolysis.Active Rho family GTPases, Rac1 and RhoA, induce the membrane translocation of downstream effectors and trigger their activation, which commonly involves post-translational modifications and conformational changes of bound proteins (, ). Membrane-bound Rac1-GTP recruits p21-activated kinases (PAKs) by binding to their Cdc42-Rac interactive binding (CRIB) domain. In resting cells, type I PAKs are localized in the cytoplasm as inactive dimers, with the regulatory domain shielding the kinase domain. Rac1 binding induces a conformational change and subsequent activation of PAKs, which then can phosphorylate downstream substrates. The PAKs’ activity converts the local activation of Rho-type GTPases into cell-wide responses (, ).Rac1 and RhoA, along with their fellow Rho GTPase family member Cdc42, work in a coordinated fashion to control cell migration (for reviews, see , , ). Rac1 is responsible for driving actin polymerization at the leading edge of a migrating cell, resulting in the formation of lamellipodia, which pushes the cell membrane forward (, , , ). Rac1 also promotes focal complex assembly (, ) and is essential for migration (href="#bib31" rid="bib31" class=" bibr popnode">Nobes and Hall, 1999). RhoA is required for cell adhesion (href="#bib31" rid="bib31" class=" bibr popnode">Nobes and Hall, 1999). It stimulates contractility in cells through myosin light-chain (MLC) phosphorylation, which induces the formation of stress fibers and focal adhesions (href="#bib9" rid="bib9" class=" bibr popnode">Chrzanowska-Wodnicka and Burridge, 1996, href="#bib39" rid="bib39" class=" bibr popnode">Ridley and Hall, 1992). From the perspective of cell morphology, Rac1 and RhoA oppose each other. Although the picture is likely more complicated (see href="#sec3" rid="sec3" class=" sec">Discussion), canonical descriptions of cell migration place active Rac1 at the migrating cell’s front and active RhoA at its back. Biochemically, Rac1 and RhoA are generally found to interact in mutually antagonistic ways, playing opposing roles in cell migration (href="#bib33" rid="bib33" class=" bibr popnode">Ohta et al., 2006, href="#bib44" rid="bib44" class=" bibr popnode">Sanz-Moreno et al., 2008; reviewed in href="#bib17" rid="bib17" class=" bibr popnode">Guilluy et al., 2011).Double-negative feedback loops resulting from mutual inhibition can lead to bistability (href="#bib21" rid="bib21" class=" bibr popnode">Kholodenko, 2006). A bistable system can flip between two biochemically distinct steady states; in the proper context, these steady states can promote different cellular phenotypes. Thus, the existence of bistability enables switch-like behaviors in which a graded, analog change in signal inputs could cause abrupt, digital responses in signaling outputs (href="#bib14" rid="bib14" class=" bibr popnode">Ferrell, 2002, href="#bib51" rid="bib51" class=" bibr popnode">Tyson et al., 2003). Bistability has been observed in many biological systems, including the mitogen-activated protein kinase (MAPK) family cascades (href="#bib2" rid="bib2" class=" bibr popnode">Bhalla et al., 2002, href="#bib25" rid="bib25" class=" bibr popnode">Markevich et al., 2004, href="#bib26" rid="bib26" class=" bibr popnode">Markevich et al., 2006, href="#bib60" rid="bib60" class=" bibr popnode">Xiong and Ferrell, 2003) and Cdc2 activation circuit (href="#bib38" rid="bib38" class=" bibr popnode">Pomerening et al., 2003, href="#bib47" rid="bib47" class=" bibr popnode">Sha et al., 2003), which play important roles in diverse cellular functions such as development and memory (href="#bib32" rid="bib32" class=" bibr popnode">Ogasawara and Kawato, 2010). Although it was suggested that mutual inhibition between Rac1 and RhoA may result in bistable activity responses (href="#bib20" rid="bib20" class=" bibr popnode">Jilkine et al., 2007, href="#bib48" rid="bib48" class=" bibr popnode">Symons and Segall, 2009, href="#bib50" rid="bib50" class=" bibr popnode">Tsyganov et al., 2012), this behavior and the consequences for cell migration have not yet been experimentally observed. Here, we combine kinetic modeling and experimentation to demonstrate the existence of bistability in the Rac1-RhoA signaling system of highly motile MDA-MB-231 cells. Model analysis and simulations predict that graded changes in PAK activity induce bistable responses of Rac1 and RhoA activities, which are experimentally validated. Furthermore, the bistable properties of the Rac1-RhoA biochemical circuitry are translated into bistability of the actin dynamics and cell migration.